ABSTRACT One important function of intracellular protein degradation is to selectively destroy misfolded or damaged proteins, as accumulate in various human diseases and during aging. Our primary goal is to understand further the functioning of the ubiquitin proteasome pathway and the involvement of molecular chaperons in this process. Most protein breakdown in mammalian cells is catalyzed by the 26S proteasome, whose 19S regulatory particle contains six ATPases which function as molecular chaperones. To understand how these ATPases unfold and translocate substrates into the core 20S proteasome, we are also studying the simpler, homologous ATPase complex, PAN, which activates proteolysis by 20S proteasomes in archaea. We recently discovered that upon ATP-binding, a conserved C-terminal sequence (HbYX motif) in these ATPases docks into pockets in the 20S's outer ring and like a "key-in-a-lock" triggers opening of its gated channel for substrate entry. A major goal will be to elucidate further the molecular events for gate-opening and its structural basis. In the eukaryotic 26S, the six ATPases and seven 20S [unreadable]-subunits differ, and gate opening is restricted to two ATPases. We hope to learn which C-termini dock into which 20S pockets and whether other proteasome regulators (BLM10, PI131) and p97/cdc48 that contain the HbYX motif activate 20S proteasomes similarly. We have dissociated proteasomal degradation into a series of ATP-requiring steps, and hope to elucidate further this multistep reaction sequence. We shall explore whether the eukaryotic 26S utilizes additional reaction steps in degrading ubiquitin-conjugated proteins. We've found that the six ATPase subunits interact through negative cooperativity and identified in PAN two substrate-binding domains whose function is nucleotide-dependent. We hope to clarify the functional significance in protein degradation of these cooperative interactions and the associated conformational changes. We recently found that increased temperatures and oxygen radicals in yeast cause the degradation specifically of newly synthesized proteins, without affecting breakdown of most cell proteins. We hope to clarify further the roles of different ubiquitination enzymes and molecular chaperones in their degradation and to identify the newly synthesized proteins most sensitive to such damage. In harsh conditions that damage cell proteins (e.g. heat-shock, oxidative stress, near-freezing temperatures), yeast produce large amounts of the "chemical chaperone", trehalose, which enhances cell viability. We recently found that trehalose is also required for efficient protein degradation, even in normal cells. The mechanisms of this unexpected new protective function of trehalose will be investigated.